US20180216071A1 - Cellular culture medium free from serum - Google Patents

Cellular culture medium free from serum Download PDF

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US20180216071A1
US20180216071A1 US15/749,183 US201615749183A US2018216071A1 US 20180216071 A1 US20180216071 A1 US 20180216071A1 US 201615749183 A US201615749183 A US 201615749183A US 2018216071 A1 US2018216071 A1 US 2018216071A1
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vitamin
aqueous solution
saline aqueous
glucose
serum
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US15/749,183
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Massimo Costa
Alberto Giuseppe Passi
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Blast Research Srl
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/16—Magnesium; Mg chelators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/92—Medium free of human- or animal-derived components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/95—Protein-free medium and culture conditions

Definitions

  • the present invention relates to a cell culture medium, especially for culturing human autologous fibroblasts intended for skin transplantation into the field of aesthetics or regenerative medicine.
  • the fibroblasts injected were able to synthesize the extracellular matrix for at least 12 months.
  • the cells exhibit an excellent cell viability and proliferation in the presence of serum and the serum generally employed is the fetal bovine serum (PBS).
  • PBS fetal bovine serum
  • This type of substances can be immunogen and therefore cause allergic reactions in some subjects.
  • Other types of cell culture media such as DMEM have extremely high amounts of glucose that can be dangerous in subjects with overt diabetes or with diabetic tendencies.
  • Tyrode's solution a physiological saline solution, called Tyrode's solution, which allows for storing organs for transplantation at low temperatures for 10-12 hours.
  • This aqueous solution consists of the following components in the respective concentrations
  • saline solution which, when employed as cell culture medium, in particular for culturing fibroblasts, maintains up to 30% of viable cells for a 24 hours' incubation period in said liquid. Also this type of saline solution allows for a markedly higher cell growth than a culture medium containing only PBS.
  • an object of the present invention is an aqueous saline solution containing
  • a further object of the present invention is a serum-free cell culture medium consisting of the above-mentioned physiological solution.
  • a further object of the present invention is a cell culture of human autologous fibroblasts in the culture medium according to the present invention for use in skin transplantation in aesthetic or regenerative medicine.
  • FIG. 1 shows in graphical presentation the MTT assay results on % cell viability of fibroblasts after 24 hours of incubation in DMEM, DMEM+PBS, PBS only, conventional Tyrode's solution, a solution according to the present invention in the presence of Vitamin C (0.1 mM) and Vitamin B6 (10 mM) and a solution according to the present invention in the presence of Vitamin C (1 mM), and Vitamin B6 (10 mM), respectively.
  • FIG. 2 shows proliferation of human fibroblasts (cell counting) after a 24 hours' incubation in the following culture media: DMEM only, DMEM+PBS, PBS only, conventional Tyrode's solution, a solution according to the present invention in the presence of Vitamin C (0.1 mM), Vitamin B6 (10 mM) and a solution according to the present invention in the presence of Vitamin C (1 mM), Vitamin B6 (10 mM).
  • the saline solution of the invention has a concentration of the component b), i.e. D-glucose, of 5.5 mM.
  • the water-soluble vitamins of the present invention are selected from at least one of the following: Vitamin C, Vitamin B12, Vitamin B6, Vitamin B5 or Pantothenic Acid, Biotin, Nicotinamide or the amide form of Vitamin B3, the salt form of ⁇ -Lipoic Acid or Vitamin N.
  • the saline solution of the invention contains at least the combination of Vitamin C and Vitamin B6.
  • Vitamin B6 it is intended pyridoxine, pyridoxamine hydrochloride, pyridoxal o pyridoxal phosphate.
  • the Vitamin C is present at a concentration between 0.05 mM and 2 mM and the Vitamin B6 is present at a concentration ranging between 5 mM and 20 mM.
  • the saline solution of the present invention consists of the following components in their respective millimolar concentrations:
  • the cells primary human dermal fibroblasts
  • the cells were grown in standard amplification conditions (37° C., 5% CO 2 , 95% humidity in the incubator) in the following culture liquids
  • Tyrode's solution modified according to the present Component invention Standard Tyrode NaCI 130 mM 137 mM KCl 2.68 mM 2.7 mM D-Glucose 5.5 mM 5.5 mM NaHCO3 11.9 mM 12 mM NaH2PO4 0.46 mM 0.2 mM MgCl2 1.05 mM 1 mM CaCl2 1.8 mM 1.8 mM Vit C 0.1 mM or 1 mM Absent Vit B6 10 mM Absent
  • the cells detached by trypsin treatment and after centrifugation and washing in sterile buffer (PBS), were suspended in the various solutions prepared and then counted with an automatic cell counter.
  • PBS sterile buffer
  • the cells were maintained in the incubator as for the amplification step.
  • the cell cultures were then assessed for viability (MTT assay) and proliferation (cell counting).
  • the two modified Tyrode's solution, object of the present invention and examined, show a significantly better proliferation compared to standard formulation containing PBS only and the standard Tyrode, although obviously lower than DMEM formulations that are markedly much more rich in amino acids and micronutrients.

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Abstract

The present invention relates to a cell culture medium, in particular for culturing autologous fibroblasts, for use in aesthetic medicine for skin transplantation, said culture medium being serum-free and being characterized in that it contains glucose in a much lower quantity than conventional culture media.

Description

    FIELD OF INVENTION
  • The present invention relates to a cell culture medium, especially for culturing human autologous fibroblasts intended for skin transplantation into the field of aesthetics or regenerative medicine.
    • BACKGROUND ART
  • In aesthetic and regenerative medicine, the use of autologous fibroblasts transplantation, in jargon also called “Autotransplantation”, is a technique employed for quite some time.
  • With aging, the number of active fibroblasts in the skin tends to decrease, as well as the metabolic capabilities thereof. The increase of cells due to transplantation of autologous fibroblasts with the ability to synthesize the extracellular matrix allows for counteracting the effects of skin aging.
  • Indeed, it was clinically demonstrated that, in patients who were injected with cultures of autologous fibroblasts from explants of the same patients and cultivated in vitro, they have improved the biomechanical properties of skin of above-mentioned patients, as demonstrated with different kinds of clinical trials, such as optical profilometry, vacuum cutometry, and the VISIA® photometric analysis.
  • Indeed, the fibroblasts injected were able to synthesize the extracellular matrix for at least 12 months.
  • In areas of the skin of the patients who have undergone treatment actually, it is noted an increase of collagen and a general thickening of dermis (up to 60% in 12 months), an increase of elasticity of the skin (25% in the periorbital area) and a general reduction of wrinkles equal to 50% (V. Zorin et al “Clinical-instrumental and morphological evaluation of the effect of autologous dermal fibroblasts administration” Journal of Tissue Engineering and Regenerative Medicine Article first published online: 19 Dec. 2014b DOI: 10.1002/term.1976).
  • Although the good results obtained with this technique, it finds it hard to take hold for a number of criticalities, first of all the cell culture medium.
  • In fact, it is well known that the culture media in which cells such as fibroblasts grows are not always suitable to be injected into humans.
  • In fact, it is known that the cells exhibit an excellent cell viability and proliferation in the presence of serum and the serum generally employed is the fetal bovine serum (PBS). This type of substances can be immunogen and therefore cause allergic reactions in some subjects. Other types of cell culture media such as DMEM have extremely high amounts of glucose that can be dangerous in subjects with overt diabetes or with diabetic tendencies.
  • It's felt the need to have cell culture media which do not present the aforesaid drawbacks, i.e. that are serum-free while containing a lower content of glucose, compared to the cell culture media such as DMEM.
  • On the other hand, it is known a physiological saline solution, called Tyrode's solution, which allows for storing organs for transplantation at low temperatures for 10-12 hours.
  • This aqueous solution consists of the following components in the respective concentrations
    • Standard Tyrode's Solution
  • NaCI 137 mM 
    KCl 2.7 mM
    D-Glucose 5.5 mM
    NaHCO3  12 mM
    NaH2PO4 0.2 mM
    MgCl
    2   1 mM
    CaCl2 1.8 mM
    • SUMMARY OF THE INVENTION
  • The applicant has now found a saline solution, which, when employed as cell culture medium, in particular for culturing fibroblasts, maintains up to 30% of viable cells for a 24 hours' incubation period in said liquid. Also this type of saline solution allows for a markedly higher cell growth than a culture medium containing only PBS.
  • Therefore, an object of the present invention is an aqueous saline solution containing
      • a) a mixture of mineral salts consisting of sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, magnesium chloride and calcium chloride,
      • b) D-glucose and
      • c) at least a water-soluble vitamin,
        wherein the concentration of b) D-glucose is <6 mM.
  • A further object of the present invention is a serum-free cell culture medium consisting of the above-mentioned physiological solution.
  • Finally, a further object of the present invention is a cell culture of human autologous fibroblasts in the culture medium according to the present invention for use in skin transplantation in aesthetic or regenerative medicine.
    • DESCRIPTION OF FIGURES
  • FIG. 1 shows in graphical presentation the MTT assay results on % cell viability of fibroblasts after 24 hours of incubation in DMEM, DMEM+PBS, PBS only, conventional Tyrode's solution, a solution according to the present invention in the presence of Vitamin C (0.1 mM) and Vitamin B6 (10 mM) and a solution according to the present invention in the presence of Vitamin C (1 mM), and Vitamin B6 (10 mM), respectively.
  • FIG. 2 shows proliferation of human fibroblasts (cell counting) after a 24 hours' incubation in the following culture media: DMEM only, DMEM+PBS, PBS only, conventional Tyrode's solution, a solution according to the present invention in the presence of Vitamin C (0.1 mM), Vitamin B6 (10 mM) and a solution according to the present invention in the presence of Vitamin C (1 mM), Vitamin B6 (10 mM).
    •  DETAILED DESCRIPTION OF THE INVENTION
  • Preferably the saline solution of the invention has a concentration of the component b), i.e. D-glucose, of 5.5 mM.
  • Preferably the water-soluble vitamins of the present invention are selected from at least one of the following: Vitamin C, Vitamin B12, Vitamin B6, Vitamin B5 or Pantothenic Acid, Biotin, Nicotinamide or the amide form of Vitamin B3, the salt form of α-Lipoic Acid or Vitamin N.
  • Preferably the saline solution of the invention contains at least the combination of Vitamin C and Vitamin B6.
  • Preferably for the objects of the present invention by Vitamin B6 it is intended pyridoxine, pyridoxamine hydrochloride, pyridoxal o pyridoxal phosphate.
  • Preferably, the Vitamin C is present at a concentration between 0.05 mM and 2 mM and the Vitamin B6 is present at a concentration ranging between 5 mM and 20 mM.
  • According to a particularly preferred embodiment of the invention, the saline solution of the present invention consists of the following components in their respective millimolar concentrations:
  •
    NaCl  130 mM,
    KCl 2.68 mM,
    D-Glucose  5.5 mM,
    NaHCO3 11.9 mM,
    NaH2PO4 0.46 mM,
    MgCl2 1.05 mM,
    CaCl2  1.8 mM,
    Vitamin C 0.1-1 mM, 
    Vitamin B6   10 mM.
  • Experimental evidences of cell viability and cellular proliferation of fibroblasts carried on cell cultures in DMEM, DMEM+PBS, only PBS, conventional Tyrode's solution, a solution according to the present invention in the presence of Vitamin C (0.1 mM), Vitamin B6 (10 mM) and a solution according to the present invention in the presence of Vitamin C (1 mM), and Vitamin B6 (10 mM), respectively, are reported for illustrative purposes herein below.
    • EXAMPLE Method
  • The cells (primary human dermal fibroblasts) were grown in standard amplification conditions (37° C., 5% CO2, 95% humidity in the incubator) in the following culture liquids
      • DMEM
      • DMEM supplemented with antibiotics and 10% serum, in the culture liquid containing PBS serum (10%)
      • Standard Tyrode
      • Two Tyrode's solutions modified according to the invention and containing each Vitamin B6 at a concentration of 10 mM and Vitamin C at a concentration of 0.1 and 1 mM, respectively.
  • Conventional or standard Tyrode's formulations and modified Tyrode's formulations according to the present invention and examined in the present example are shown in the following table herein below.
  • Tyrode's solution modified
    according to the present
    Component invention Standard Tyrode
    NaCI  130 mM 137 mM 
    KCl 2.68 mM 2.7 mM
    D-Glucose  5.5 mM 5.5 mM
    NaHCO3 11.9 mM  12 mM
    NaH2PO4 0.46 mM 0.2 mM
    MgCl2 1.05 mM   1 mM
    CaCl2  1.8 mM 1.8 mM
    Vit C 0.1 mM or 1 mM Absent
    Vit B6   10 mM Absent
  • At the time of the experiment, the cells, detached by trypsin treatment and after centrifugation and washing in sterile buffer (PBS), were suspended in the various solutions prepared and then counted with an automatic cell counter.
  • During the 24 h incubation, the cells were maintained in the incubator as for the amplification step.
  • The cell cultures were then assessed for viability (MTT assay) and proliferation (cell counting).
  • The results of the above tests are reported in FIGS. 1 and 2, respectively.
  • As can be seen after a 24 hours' incubation in serum-free media (PBS and Tyrode's standard) the cells have in fact completely lost their viability. In the case of culture medium DMEM with serum, the cells have more than 90% of survival that drops to just over 60% in DMEM without serum, however in the presence of high doses of amino acids, vitamins, and 25 mM glucose, which is five times the standard concentration. The modified Tyrode's solutions according to the present invention instead maintain the cells viable up to 30% after 24 hours, a much better result than the standard Tyrode's solution, but also than the culture liquid containing PBS only.
  • The two modified Tyrode's solution, object of the present invention and examined, show a significantly better proliferation compared to standard formulation containing PBS only and the standard Tyrode, although obviously lower than DMEM formulations that are markedly much more rich in amino acids and micronutrients.

Claims (9)

1. A saline aqueous solution comprising:
a) a mixture of biologically acceptable mineral salts consisting of sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, magnesium chloride and calcium chloride,
b) D-glucose and
c) at least a water-soluble vitamin, wherein the concentration of b) glucose is <6 mM.
2. The saline aqueous solution according to claim 1, wherein the concentration of b) glucose is 5.5 mM.
3. The saline aqueous solution according to claim 1, wherein said water-soluble vitamins are selected from at least one of the following: Vitamin C, Vitamin B12, Vitamin B6, Vitamin B5, Biotin, Nicotinamide, salt form of α-Lipoic Acid.
4. The saline aqueous solution according to claim 1, containing at least vitamin C and vitamin B6.
5. The saline aqueous solution according to claim 4, wherein said Vitamin C is present at a concentration between 0.05 mM and 2 mM and said Vitamin B6 is present at concentration between 5 mM and 20 mM.
6. The saline aqueous solution according to claim 4, characterized in that wherein said solution is a modified Tyrode's solution and consists of the following components in the respective millimolar concentrations:
NaCl  130 mM, KCl 2.68 mM, D-Glucose  5.5 mM, NaHCO3 11.9 mM, NaH2PO4 0.46 mM, MgCl2 1.05 mM, CaCl2  1.8 mM, Vitamin C 0.1-1 mM,  Vitamin B6   10 mM.
7. A serum-free cell culture medium consisting of the saline aqueous solution according to claim 1.
8. A cell culture of fibroblasts expanded in the serum-free cell culture medium according to claim 7.
9. The cell culture according to claim 8, wherein said fibroblasts are human autologous fibroblasts for use in skin transplantation in aesthetic medicine.
US15/749,183 2015-08-03 2016-07-28 Cellular culture medium free from serum Abandoned US20180216071A1 (en)

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US6103529A (en) * 1996-10-10 2000-08-15 Life Technologies, Inc. Animal cell culture media comprising peptides derived from rice
JP4916706B2 (en) * 2004-11-10 2012-04-18 株式会社機能性ペプチド研究所 Completely synthetic medium for mammalian fibroblasts
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US20160108361A1 (en) * 2013-04-17 2016-04-21 Salk Institute For Biological Studies Media compositions for neuronal cell culture

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